7-t-butoxyiminomethylcamptothecin conjugated in position 20 with integrin antagonists

ABSTRACT

Compounds of Formula (I) are described: 
                         
in which the R 1  group is as defined in the specification and includes the condensation of 7-t-butoxyiminomethylcamptothecin in position 20 with a cyclopeptide containing the RGD sequence. Said compounds are endowed both with high affinity for integrin receptors α v β 3  and α v β 5  and with selective cytotoxic activity on human tumour cell lines at micromolar concentrations.

This application is the US national phase of international applicationPCT/IT2005/000245, filed 28 Apr. 2005, which designated the U.S. andclaims priorityof IT RM2004A000242, filed 13 May 2004, the entirecontents of each of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compounds with cytotoxic activityconsisting of cyclopeptides containing the RGD sequence and derivativesof camptothecin, methods for the preparation thereof, their use asmedicaments and compositions containing them.

In particular, the compounds described in the present invention areendowed with both high affinity for integrins α_(v)β₃ and α_(v)β₅ andselective cytotoxic activity on human cell lines at micromolarconcentrations.

BACKGROUND TO THE INVENTION

Chemotherapeutic anticancer agents are the drugs with the mostrestrictive therapeutic window. In fact, since their cytotoxic activityis non-selective they may indiscriminately damage all the cells of thebody with which they come into contact.

There currently exists the problem of directing the cytotoxic agentselectively against the tumour cells, allowing the agent to exert itsactivity without damaging the cells of the healthy surrounding tissues,or at least limiting the damage as much as possible.

It has been reported in the literature that blocking the integrinsα_(v)β₃ and α_(v)β₅ by means of the use of selective cyclopeptides, thereference compound for which is regarded as cyclopentapeptidec(Arg-Gly-Asp-D-Phe-Val) (JACS 1997, 119, 1328-35; international patentapplication WO 97/06791), or by means of the use of monoclonalantibodies (Cell, 1994, 79, 1157-64) leads to the blocking ofangiogenesis and to a reduction of tumour growth. In addition,antimetastatic effects have also been observed (J. Clin. Invest., 1995,96, 1815). Brooks et al. (Science, 1994, 264, 569-71) reported that theendo-thelial cells of the tumour vasculature and the tumour cellsthemselves preferentially express integrin α_(v)β₃ compared to thequiescent cells of normal tissue. Among the compounds at an advancedstage of clinical development, we may mentionc(Arg-Gly-Asp-D-Phe-MeVal), or EMD121974 or cilengitide.

Ruoslati and co-workers (Current Opinion in Oncology, 1998, 10, 560-5)showed that RGD analogues that bind to the tumour endothelium, onceconjugated to the cytotoxic agent doxorubicin, form compounds that aremore efficient and less toxic than doxorubicin alone. These authors alsodemonstrated, beyond any reasonable doubt, that the effect isattributable to the conjugation to RGD, inasmuch as the binding isantagonised by the free peptide itself (Arap, Pasqualini and Ruoslati,Science, 1998, 279, 377-380). Later, the same authors carried out otherexperiments consisting in chemically binding a pro-apoptotic peptidesequence to an RGD analogue, demonstrating that the new compounds wereselectively toxic for angiogenic endothelial cells and had anticanceractivity in mice (Ruoslati, Nature Medicine, 1999, 5, 1032-8).

Marcus et al., in international patent application WO 01/17563, describespecific anticancer activity for cytotoxic agents, such as camptothecin,conjugated by means of a spacer, consisting of one or more amino acids,to a non-peptidic inhibitor antagonist of integrins α_(v)β₃ and α_(v)β₅.

Aoki et al., Cancer Gene Therapy, 2001, 8, 783-787 describe the specificanticancer activity of a histidylated oligolysine conjugated to an RGDsequence, revealing a homing effect for tumours in mice.

The concept of binding at the cell surface mediated by integrins hasbeen proposed for gene transport (Hart, et al., J. Biol. Chem., 1994,269, 12468-12474).

7-tert-butoxyiminomethylcamptothecin (or CPT184 or ST 1481 or Gimatecan)is a derivative of camptothecin which is active orally and is describedin European Patent EP 1 044 977.

It has now been found that 7-t-butoxyiminomethylcamptothecin conjugatedin position 20, possibly by means of suitable spacers, to cyclopeptidederivatives containing the RGD sequence yields compounds endowed withhigh, selective anticancer activity which can be advantageously used forthe preparation of medicaments for the treatment of tumours.

By virtue of their selective cytotoxic activity on tumour cells, thecompounds according to the present invention yield medicaments withfewer and less severe side effects.

DESCRIPTION OF THE INVENTION

The object of the present invention are derivatives of7-t-butoxyiminomethylcamptothecin conjugated to cyclopeptide derivativescontaining the RGD sequence. The resulting molecules possess unalteredboth the cytotoxic properties of the original camptothecins and integrinbinding properties with affinity comparable to that observed with thenon-conjugated cyclopeptides. The result of this combination is tofavour the concentration of the cytotoxic agent in those cells that mostexpress integrins of the α_(v)β₃ and α_(v)β₆ type (homing). Thecytotoxic agent exerts its intracellular activity in the conjugatedand/or free form through enzymatic or hydrolytic action. The main objectof the present invention are therefore compounds of Formula (I)

-   -   where:    -   R₁ is the U-X-Y group, in which:    -   U is either absent or one of the following groups —COCHR₁₀NH— or        CON[(CH₂)_(n2)NHR₇]—CH₂—, where R₁₀ is H or is selected from the        group consisting of: linear or branched C₁-C₄ alkyl, optionally        substituted with C₆-C₁₄ aryl or an amino-alkyl C₁-C₄; R₇ is H or        linear or branched C₁-C₄ alkyl; n₂ is an integer number from 2        to 6;    -   X is absent or is H or is a group selected among the following:        —COCHR₃NH—, —COCHR₆(CH₂)_(n3)R₄—, —R₄—CH₂(OCH₂CH₂)_(n4)OCH₂R₄—,        —R₄(Q)R₄—, —R₅[Arg-NH(CH₂)_(n5)CO]_(n6)R₅—,        —R₅-[N-guanidinopropyl-Gly]_(n6)R₅—, in which n₃ is an integer        number from 0 to 5, n₄ is an integer number from 0 to 50, n₅ is        an integer number from 2 to 6, n₆ is an integer number from 2 to        7;    -   R₃ is H or linear or branched C₁-C₄ alkyl, optionally        substituted with —COOH, —CONH₂, —NH₂ or —OH;    -   R₄ is selected from the group consisting of: —NH—, —CO—, —CONH—,        —NHCO—;    -   R₅ is either absent or is the group —R₄(Q)R₄—;    -   R₆ is H or NH₂;    -   Q is selected from the group consisting of: linear or branched        C₁-C₆ alkylene; linear or branched C₃-C₁₀ cycloalkylene; linear        or branched C₂-C₆ alkenylene; linear or branched C₃-C₁₀        cyclo-alkenylene; C₆-C₁₄ arylene; arylene (C₆-C₁₄)-alkylene;        (C₁-C₆), alkylene (C₁-C₆)-arylene (C₆-C₁₄); aromatic or        non-aromatic heterocyclyl (C₃-C₁₄), containing at least one        heteroatom selected from the group consisting of O, N, S;    -   Y is absent or H or is the following group        c(Arg-Gly-Asp-AA₁-AA₂),    -   in which:    -   c means cyclic;    -   AA₁ is selected from the group consisting of: (D)-Phe, (D)-Trp,        (D)-Tyr, (D)-2-naphthylAla, (D)-4-terbutyl-Phe,        (D)-4,4′-biphenyl-Ala, (D)-4-CF₃-Phe, (D)-4-acetylamino-Phe;    -   AA₂ is selected from the group consisting of:        NW—CH[(CH₂)_(n7)—CO]—CO, NW—CH[(CH₂)_(n7)—NH]—CO,        NW-[4-(CH₂)_(n7)—CO]-Phe, NW-[4-(CH₂)_(n7)—NH]-Phe, [NW]-Gly,        NW-Val, in which W is selected from H, linear or branched C₁-C₆        alkyl, —(CH₂)_(n7)—COOH where n₇ is an integer number from 0 to        5, 4-carboxybenzyl, 4-aminomethylbenzyl;    -   with the proviso that X and Y cannot be both absent; the        N₁-oxides, racemic mixtures, their single enantiomers, their        single diastereoisomers, the forms E and Z, mixtures thereof,        the pharmaceutically acceptable salts.

The present invention comprises the use of compounds with theabove-mentioned compounds of Formula (I) as active ingredients formedicaments useful as topoisomerase I inhibitors. Among the therapeuticapplications deriving from topoisomerase I inhibition, we mentionparasitic or viral infections

Given their particular pharmacological characteristics, the compounds ofFormula (I) compounds are also useful for the preparation of medicamentsfor the treatment of tumours and the metastatic forms thereof.

The present invention also comprises pharmaceutical compositionscontaining compounds of Formula (I) as active ingredients, in mixtureswith at least one pharmaceutically acceptable vehicle and/or excipient.

The compounds according to the present invention are the result of thecondensation of 7-t-butoxyiminomethylcamptothecin, functionalised inposition 20, with a cyclopeptide containing the Arg-Gly-Asp (RGD)sequence. This structural combination has the advantage of favouring theconcentration of the cytotoxic agent (camptothecin) in the cells thatmost express integrins of the α_(v)β₃ and α_(v)β₅ type. The cytotoxicagent exerts its activity in the conjugated and/or free form throughenzymatic or hydrolytic action.

The definitions of the various functional groups and residues, as wellas the definitions of the pharmaceutically acceptable salts that figurein the above-mentioned formula (I), are common knowledge to any expertchemist and no particular definitions are necessary. However, referenceto such groups can be found in the technical and patent literature, e.g.in international patent applications WO 00/53607, WO 03/101995 and WO03/101996.

One initial group of preferred compounds consists of the compounds ofFormula (I) where U and/or X are not absent.

The preferred compounds according to the present invention are thefollowing:

Compounds of Formula (I) can be prepared with the process described herebelow and exemplified for the preferred compounds according to theinvention. This process constitutes a further object of the invention.

Fundamentally, the compounds of Formula (I) which are the object of thepresent invention are prepared by means of the condensation of7-t-butoxyiminomethylcamptothecin (indicated as “7-t-but-CP”), possiblyfunctionalised via a suitable bridge (indicated as “U₁-X₁”) , with acyclopeptide derivative (indicated as “Y₁”).

The condensation reactions can be carried out according to one of thefollowing reaction schemes:7-t-but-CP+U₁-X₁-Y₁ or7-t-but-CP-U₁+X₁-Y₁ or7-t-but-CP-U₁-X₁+Y₁ or7-t-but-CP+U₁+X₁+Y₁ or7-t-but-CP-U₁+X₁+Y₁;where 7-t-but-CP represents 7-t-butoxyiminomethylcamptothecin, U₁, X₁and Y₁ represent respectively the groups U, X and Y as defined inFormula I, eventually appropriately functionalised and/or protected sothat the conjugated compounds of Formula I are obtained.

These reactions are conducted using conventional methods, such as, forinstance, those described Journal of Controlled Release 2003, 91, 61-73;S. S. Dharap et al.; Journal of Medicinal Chem. 2003, 46, 190-3, R.Bhatt;

The cyclopeptides Y₁ can be prepared according to conventional peptidesynthesis techniques, as described in examples 1 to 6. The peptidesynthesis can be accomplished either in the solid phase or in solution.

Once the desired cyclopeptide has been obtained, it will be used in thecondensation reaction in its protected form, and the protector groupswill be removed only after obtaining the final compound. Thedeprotection is done using known methods, e.g. acid conditions by meansof the use of pure trifluoroacetic acid or in the presence ofchlorinated organic solvents.

The compounds described in the present invention are topoisomerase Iinhibitors and are therefore useful as medicaments, particularly for thetreatment of diseases that benefit from the inhibition of saidtopoisomerase. In particular, the compounds according to the presentinvention exhibit antiproliferative activity, and therefore are used fortheir therapeutic properties, and possess physicochemical propertieswhich make them suitable for formulation in pharmaceutical compositions.

The pharmaceutical compositions contain at least one formula (I)compound as an active ingredient, in an amount such as to produce asignificant therapeutic effect. The compositions covered by the presentinvention are entirely conventional and are obtained using methods thatare common practice in the pharmaceutical industry. According to theadministration route opted for, the compositions will be in solid orliquid form and suitable for oral, parenteral or intravenousadministration. The compositions according to the present inventioncontain, along with the active ingredient, at least one pharmaceuticallyacceptable vehicle or excipient. Formulation adjuvants, such as, forexample, solubilising agents, dispersing agents, suspension agents oremulsifying agents may be particularly useful.

The compounds of Formula (I) can also be used in combination with otheractive ingredients, such as, for example, anticancer agents or otherdrugs with antiparasitic or antiviral activity, both in separate formsand in a single dosage form.

The compounds according to the present invention are useful asmedicaments with anticancer activity, e.g. in non-microcytoma andsmall-cell lung cancer, or in colorectal or prostate cancer,glioblastoma and neuroblastoma, cervical cancer, ovarian cancer,gastrointestinal carcinoma, carcinoma of the liver, Kaposi's sarcoma,renal carcinoma, sarcoma and osteosarcoma, testicular carcinoma,carcinoma of the breast, carcinoma of the pancreas, melanoma, carcinomaof the urinary bladder and of the head and neck. One of the advantagesafforded by the compounds according to the present invention is thecombination of antitopoisomerase activity, proper to the camptothecinportion of the molecule, and the integrin inhibiting activity, providedby the cyclopeptide portion of the molecule. The result is the possiblecombined action of the compounds according to the present inventionwhich will be favourably received in the oncological sector by theexperts operating in that sector. In fact, the cyclopeptide portion,containing the Arg-Gly-Asp sequence, not only directs the moleculeagainst tumours expressing integrins, but, once the target has beenreached, is capable of exerting multiple functions, ranging from theinternalisation of the cytotoxic portion of the molecule to integrininhibiting activity, with the resulting advantages, particularly interms of the inhibition of tumour angiogenesis. The cyclopeptideportion, once separated from the camptothecin portion, is also capableof exerting its action at a distance from the site of the tumour, andtherefore the compounds according to the present invention also proveuseful in the prevention or treatment of metastatic forms.

The medicaments which are the object of the present invention can alsobe used in the treatment of parasite diseases.

The following examples further illustrate the invention.

The abbreviations used are the following:

-   Aad (aminoadipic acid);-   Amb (aminomethylbenzyl);-   Amp (aminomethylphenylalanine);-   Boc (tert-butoxycarbonyl);-   CSA (camphosulfonic acid);-   CTH (catalytic transfer hydrogenation);-   DCC (dicyclohexylcarbodiimide);-   DCM (dichloromethane);-   DIEA (diisopropylethylamine);-   DMF (dimethylformamide);-   Dy(OTf)₃ dysprosium triflate;-   Fmoc (9-fluorenylmethyloxycarbonyl);-   HOBT (hydroxybenzotriazole);-   NMP (N-methyl-pyrrolidone);-   Pht (phthaloyl);-   Pmc (pentamethylchroman-6-sulphonyl);-   ST1481 (7-t-butoxyiminomethylcamptothecin, also named gimatecan)-   TBTU (tetrafluoroborate-O-benzotriazol-1-yl-tetramethyluronium);-   TFA (trifluoroacetic acid).

EXAMPLES Example 1 Synthesis of c(Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-Amp)(Protected ST2581)

1.587 mmol of Fmoc-Gly-Res (Res=Sasrin Resin@, Bachem) were suspendedunder stirring in 75 ml of DMF for 30 minutes, after which 18 ml ofpiperidine were added, continuing the stirring for a further 30 minutes.The resin, filtered and washed with DMF, was suspended in 50 ml of NMP(N-methyl-pyrrolidone) for 15 minutes, after which Fmoc-Arg(Pmc)-OH,HOBT, TBTU and DIEA were added (3.174 mmol of each); after 2 hours ofstirring, the suspension was filtered and washed with DMF. Afterdeprotection with piperidine, the coupling was repeated with the otheramino acids in succession, operating each time as described above,namely: Fmoc-Amp(Cbz)-OH, Fmoc-D-Phe-OH, and Fmoc-Asp(OtBu)-OH. Afterthe last deprotection of the Fmoc-N-terminal, the linear pentapeptidewas released from the resin with 45 ml of 1% TFA in DCM. This wasdissolved in approximately 11 of CH₃CN, and 4.761 mmol of HOBT and TBTUand 10 ml of DIEA were added; the solution was kept under stirring for30 minutes, the solvent was evaporated to a small volume and theprecipitation of the product was completed with water. The filteredcrude product was dissolved in 27 ml of a mixture of MeOH and DMF 1:1; 5mmol of ammonium formiate and 0.55 g of 10% Pd/C were added and leftunder stirring at room temperature for 30 minutes. The suspension wasfiltered on celite and brought to dryness. The residue was purified bypreparatory RP-HPLC (column: Alltima®C-18, Alltech; mobile phase 50%CH₃CN in water +0.1% TFA; retention time (Rt)=9.13 minutes). 483 mg of awhite powder were obtained.

¹H-NMR (DMSO-d₆)δ8.3, 8.07, 8.04, 7.90, 7.80, 7.33, 7.15, 7.07, 4.62,4.50, 4.35, 4.12, 4.01, 3.15, 3.03, 2.96-2.65, 2.58, 2.48, 2.32, 2.02,1.75, 1.50, 1.35, 1.23. Molecular mass (Maldi-Tof): 973

Example 2 Synthesis of c(Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-Aad) (protectedST2650)

0.69 mmol of Fmoc-Gly-Res were treated exactly as described in example1, with the difference that in this case the third and fourth aminoacids were added in the form of dipeptide Fmoc-D-Phe-Aad(OBzl)-OH. Afterdeprotection by means of CTH, and purification of the crude product withpreparatory RP-HPLC (mobile phase: 66% CH₃CN in water+0.1% TFA; Rt=17.29minutes), 187 mg of pure peptide were obtained.

¹H-NMR (DMSO-d₆)δ 7.23, 4.58, 4.20-3.90, 3.28, 3.05, 2.99, 2.85,2.74-2.35, 2.15, 2.05, 1.85-1.25. Molecular mass (Maldi-Tof): 940

Example 3 Synthesis of c(Arg(Pmc)-Glv-Asp (OtBu)-D-Phe-N-Me-Amp)(Protected ST2700)

To a suspension of Fmoc-Phe(4-Pht-N-CH₂)—COOH in anhydrous toluenebrought to reflux 2 eq of CSA and 20 eq of paraformaldehyde were added,divided into 4 portions at intervals of 15 minutes. The mixture was leftto cool, diluted with 120 ml of toluene and washed with 5% NaHCO₃ andwater. After evaporation of the solvent, the residue was dissolved in 15ml of CHCL₃+15 ml of TFA+700 μl of Et₃SiH; the mixture was left in thedark to stir for 42 hours. After evaporation of the solvent, the residuewas purified by filtration on silica gel. Overall yield: 90%.

The linear peptide was synthesized in solid phase as described inexample 1, inserting Fmoc-N-Me-Phe-(4-Pht-N-CH₂)—COOH as the third aminoacid, prepared as described above. In this case the deprotections ofN-Fmoc-terminal on resin were carried out with 30% diisopropylamine (300eq) in solution in DMF (owing to the presence of phthalimide). Aftercyclisation, 500 mg of the peptide were dissolved hot in 10 ml ofabsolute EtOH, to which 0.9 ml of a solution of NH₂-NH₂.H₂O 1 M inethanol was added. After heating at reflux for 2 hours, the solvent wasevaporated and the residue taken up with 10 ml of DCM+10 ml of Na₂CO₃solution under vigorous shaking. The crude final product was recoveredfrom the organic phase after evaporation and purified by preparatoryRP-HPLC (mobile phase: 52% CH₃CN in water+0.1% TFA; Rt=10 minutes).

¹H-NMR (CDCl₃)δ 8.29-7.66, 7.38-7.07, 4.95-4.77, 4.09, 3.41, 3.05-2.81,2.51, 2.05, 1.74, 1.40, 1.26. Molecular mass (Maldi-Tof): 987

Example 4 Synthesis ofc[Arg(Pmc)-Gly-Asp-(OtBu)-D-Phe-Amp(CO—(CH₂)₂—COOH)](Protected ST2649)

120 mg of cyclopeptide c[Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-Amp]·TFA (preparedas described in example 1) were dissolved in 3.6 ml of a mixture ofDCM-DMF 2:1, together with a stoichiometric amount of TEA and succinicanhydride. After 1 hour the reaction mixture was diluted with 30 ml ofDCM and washed with water. The organic phase, dried and concentrated,yielded a residue of 100 mg of pure product.

Analytical RP-HPLC: column: Purosphere STAR®, Merck; mobile phase: 45%CH₃CN in water+0.1% TFA; Rt=13.17 minutes.

¹H-NMR(DMSO-d₆)δ8.20-7.75, 7.19-7.02, 4.58, 4.45, 4.36, 4.30, 4.20,4.05, 3.00, 2.97-2.57, 1.83, 1.62, 1.32. Molecular mass (Maldi-Tof):1073

Example 5 Synthesis of c(Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-N-Amb-Gly)(Protected ST2701)

To a solution of 1-22 mmol of Boc-monoprotected p-xylylenediamine in 6ml of THF were added 1.83 mmol of TEA and, dropwise, a solution of 1.22mmol of benzyl bromoacetate in 2 ml of THF. The mixture was left understirring overnight, after which the solvent was evaporated and theresidue purified on a flash column (CHCl₃-EtOAc, 9:1). 0.69 mmol ofN-(4-Boc-NH-CH₂-benzyl)-glycine benzylester were obtained.

250 mg of Fmoc-D-Phe-OH were dissolved in 27 ml of DCM and 40 μl ofdiphosgene and 230 μl of sym-collidine were added; after 15 minutes 190mg of the previously prepared ester were added, dissolved in 3 ml ofDCM. After 3 hours, 80 μl of N-Me-piperazine were added to the reactionmixture and stirred for 10 minutes, after which the mixture was dilutedwith 10 ml of DCM and extraction was done with water, HCl 0.5N, water,5% NaHCO₃ and water. After evaporation of the solvent, the residue waspurified by flash chromatography on silica gel (DCM-EtOAc, 9:1). Yield:80%.

To 100 mg of the product thus obtained, dissolved in 6 ml of MeOH, wereadded 76 μl of AcOH and 42 mg of HCOONH₄, and the mixture cooled to 0°C., and 50 mg of 10% Pd/C were added. After 30 minutes, the reactionmixture was filtered on celite. The filtrate was brought to dryness andpurified on a flash column (CHCl₃-MeOH 9:1). Yield: 90%.

190 mg of the product thus obtained were dissolved in 1.2 ml of TFA andbrought to dryness (deprotection of Boc); the residue was redisolved in9 ml of 10% Na₂CO₃+6 ml of dioxane, cooled to 0° C. and a solution of120 μl of benzyloxycarbonyl chloride diluted with 3 ml of dioxane wasadded dropwise. After 1 hour stirring at room temperature, evaporationwas carried out under vacuum to a small volume, after which the mixturewas diluted with water, the pH was reduced to 1 with HCl and extractionwas done with EtOAc. After evaporation of the solvent, the residue waspurified by filtration on silica gel, washing with CHCl₃-MeOH (8:2).Pure dipeptide yield: 82%.

0.69 mmol of Fmoc-Gly-Res were treated as described in example 1. AfterArg, the previously prepared dipeptideFmoc-D-Phe-N(4-Cbz-NH—CH₂-benzyl)-Gl was added in sequence. Afterdeprotection of Cbz by means of CTH, the crude productc(Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-N-Amp-Gly) was purified by preparatoryRP-HPLC (mobile phase: 50% CH₃CN in water+0.1% TFA; Rt=10.5 minutes).

¹H-NMR (DMSO-d₆)δ8.29-7.66, 7.44-6.90, 5.15, 4.72-4.18, 4.20, 4.05-3.32,3.15, 3.06, 2.70, 2.51, 2.49, 2.01, 1.80-1.35, 1.49, 1.35, 1.23.Molecular mass (Maldi-Tof): 973

Example 6 Synthesis ofc(Arg(Pmc)-Glv-Asp(OtBu)-D-Phe-Amp(CO—CH₂—(OCH₂CH₂)_(n)—O—CH₂—COOH))

To a solution of 200 mg of c(Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-Amp)·TFA(obtained as described in example 1) in 4 ml of a 3:1 DCM-DMF mixturewas added a substantial excess of glycol diacid. DIEA (3 eq) and DCC (2eq) were added to the same solution. The mixture was left under stirringovernight, after which it was diluted with DCM and washed with water.

The crude product was recovered by evaporating the organic phase andpurified by flash chromatography (mobile phase: CHCl₃-MeOH 7:3+1% AcOH);the fractions containing the product were pooled, washed with water,dehydrated and brought to dryness, and yielded a residue of 157 mg ofpure product.

Analytical RP-HPLC: (column: Purosphere STAR®, Merck; mobile phase: 50%CH₃CN 50% in water+0.1% TFA; R_(t)=10.96)

¹H-NMR (DMSO-d₆)δ8.35-7.92, 7.20-7.00, 4.65, 4.50, 3.94, 3.60-3.45,3.00-2.60, 2.55, 2.45, 2.30, 2.00, 1.70, 1.50, 1.30, 1.20. Molecularmass (Maldi-Tof): corresponding to the different glycols used of variousmolecular weights.

Synthesis of Gimatecan Derivatives

Example 7 Synthesis of 20-O-Val-gimatecan-ST2678

One mmol of ST1481 prepared as described in Example 2 of patent EP 1 044977, 0.6 mmol of Dy(OTf)₃, 3 mmol of dimethylaminopyridine and 3 mmol ofBoc-Val-OH were suspended in 15 ml of anhydrous CH₂Cl₂ and brought to−10° C.; after 30 minutes 3.1 mmol of DCC were added and after another30 minutes at −10° C. the reaction mixture was heated to roomtemperature. After 2 hours the reaction was diluted with another 20 mlof CH₂Cl₂, washed with 1N HCl, with NaHCO₃ and dried on Na₂SO₄. Thecrude product was purified by chromatography on SiO₂ with CH₂Cl₂/MeOH97:3 to give the product as a yellow solid with a yield of 92%. Rf=0.72in CH₂Cl₂/MeOH 96:4.

Analytical RP-HPLC: (column: Luna C18, Phenomenex®; mobile phase: 45%CH₃CN in water; Rt=23.0)

¹H-NMR (CDCl₃)δ9.05, 8.3-8.2, 7.9-7.7, 7.3, 5.8-5.7, 5.5-5.4, 5.05-4.95,4.4-4.3, 2.4-2.2, 1.6-1.4, 1.1-0.9. Molecular mass (ESI): 646

The intermediate product ST2678 [N-Boc] is deprotected in DCM/TFA(75/25) at 0° C., with a quantitative yield. The ST 2678 thus obtainedcan be used to bind RGD derivatives directly or as a furtherintermediate, which can be used to bind a second residue (see examples8-9).

Example 8 Synthesis of 20-O-Val-Asp-gimatecan-ST2676 [N-Boc]

One mmol of ST2678 and 3.7 mmol of DIPEA were added in that order to asolution of 1.2 mmol of suitably protected aspartic acid, 1.8 mmol ofHOBt and 1.4 mmol of EDC in DMF at 0° C. The reaction mixture was leftovernight at room temperature before being partitioned between water anddichloromethane, and the crude product thus obtained was purified bychromatography on SiO₂ with CH₂Cl₂/MeOH 96:4 to give the product as ayellow solid with a yield of 66%. Rf=0.5 in CH₂Cl₂/MeOH 96:4

Analytical RP-HPLC: (column: Luna C18, Phenomenex®; mobile phase: 45%CH₃CN in water; Rt=23.3).

¹H-NMR (CDCL₃)δ9.05, 8.3-8.2, 7.9-7.7., 7.4-7.2, 6.0-5.9, 5.7-5.6,5.5-5.4, 5.1, 4.8-4.7, 4.6-4.5, 3.3-3.1, 3.0-2.8, 2.4-2.2, 1.6-1.4,1.-0.9. Molecular mass: (ESI): 851

Deprotection of the Carboxyl Group

The benzylester was hydrogenolysed with H₂/10%Pd-C at 20 psi with ayield of 70% after purification with CH₂Cl₂/MeOH 94:6. Rf=0.52 inCH₂Cl₂/MeOH 92:8.

Analytical RP-HPLC: (column: Luna C18, Phenomenex®; mobile phase: 45%CH₃CN in water; Rt=22.9)

¹H-NMR (CDCl₃)δ9.05, 8.3-8.2, 7.9-7.7, 7.5, 6.0-5.9, 5.7-5.6, 5.5-5.4,4.8-4.7, 4.5-4.4, 3.3-3.1, 2.9-2.8, 2.4-2.2, 1.6-1.4, 1.1-0.9. Molecularmass (ESI): 761

Example 9 Synthesis of Compound [ST2677]

One mmol of deprotected ST2678-[N-Boc] was dissolved in 10 ml ofanhydrous pyridine and after bringing the solution to 0° C., 2.5 mmol ofsuccinic anhydride were added: the mixture was restored to roomtemperature for 1 hour. The solvent was removed, the residue was takenup with CH₂Cl₂ and the organic phase was washed with 0.5N HCl. The crudeproduct was purified by chromatography on SiO₂ with CH₂Cl₂/MeOH 95:5 togive the expected product as a yellow solid with a yield of 90%. Rf=0.41in CH₂Cl₂/MeOH 92:8.

Analytical RP-HPLC: (column; Luna C18, Phenomenex®; mobile phase: 45%CH₃CN in water; Rt=17.0)

¹H-NMR (CDCl₃)δ9.05, 8.4-8.2, 7.9-7.7, 7.5, 6.4-6.3, 5.7-5.6, 5.5-5.4,4.6-4.5, 3.7, 3.0-2.1, 1.5, 1.1-0.9. Molecular mass (ESI): 646

Synthesis of Conjugated Derivatives

Example 10 Synthesis of Compounds ST2670 (or ST2671)

The same synthesis process was used for both.

To a solution of 1.2 mmol of ST2676 [N-Boc] (or ST 2677) in anhydrousDMF cooled to 0° C. were added 2.1 mmol of HOBt and 1.4 mmol of EDC andthe resulting mixture was stirred for 30 minutes before adding 1 mmol ofST2581 and DIPEA in sequence. After being left overnight the mixture waspartitioned between water and dichloromethane and the organic phase wasthen dried on Na₂SO₄ and the crude product purified by chromatography onSiO₂ with CH₂Cl₂/MeOH 92:8 to give the expected product, protectedST2670 (or protected ST2671), with a yield ranging from 55% to 65%.

Analytical RP-HPLC: (column: Luna C18, Phenomenex®; mobile phase: 45%CH₃CN in water; Rt=20.1 for protected ST2670 and Rt=23.2 for protectedST2671) Molecular mass (ESI): 1718 for protected ST2671Molecular mass(ESI): 1601 for protected ST2670

Deprotection of Conjugated Products

The final deprotection to obtain the two compounds ST2670 and ST2671 wasdone for both compounds with CH₂Cl₂/TFA 1:1 for 2 hours bringing themixture from 0° C. to room temperature; this operation was followed by astep on ion-exchange resin which gave the products as hydrochlorides.

Analytical RP-HPLC: (column: Luna C18, Phenomenex®; mobile phase: 35%CH₃CN in water; Rt=14.5 for ST2670 and Rt=14.3 for ST2671) Molecularmass (ESI): 1294 for ST2671

Molecular mass (ESI): 1279 for ST2670Biological ResultsBinding to Integrin α_(v)β₃ Receptors

The purified α_(v)β₃ receptor (Chemicon, cat. CC1020) was diluted inbuffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mMMnCl₂) at a concentration of 0.5 μg/ml. An aliquot of 100 μl was addedto 96-well plates and incubated overnight at +4° C. Plates were washedonce with buffer (50 mM Tris, pH 7,4, 100 mM NaCl, 2 mM CaCl₂, 1 mMMgCl₂, 1 mM MnCl₂, 1% bovine serum albumin) and then incubated foranother 2 hours at room temperature. Plates were washed twice with thesame buffer and incubated for 3 hours at room temperature with theradioactive ligand [¹²⁵I]echistatin (Amersham Pharmacia Biotech) 0.05 nMin the presence of competition ligands. At the end of incubation, thewells were washed and the radioactivity determined using a gamma counter(Packard). Non-specific binding of the ligand was determined in thepresence of excess cold echistatin (1 μM).

Binding to Integrin α_(v)β₅ Receptors

The purified α_(v)β₅ receptor (Chemicon, cat. CC1020) was diluted inbuffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mMMnCl₂) at a concentration of 1 μg/ml. An aliquot of 100 μl was added to96-well plates and incubated overnight at +4° C. Plates were washed oncewith buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1mM MnCl₂, 1% bovine serum albumin) and then incubated for another 2hours at room temperature. Plates were washed twice with the same bufferand incubated for 3 hours at room temperature with the radioactiveligand [¹²⁵I]echistatin (Amersham Pharmacia Biotech) 0.15 nM in thepresence of competition ligands. At the end of incubation, the wellswere washed and the radioactivity determined using a gamma counter(Packard). Non-specific ligand binding was determined in the presence ofexcess cold echistatin (1 μM).

Evaluation of IC₅₀ Parameters

The affinity of the products for vitronectin receptors was expressed asIC₅₀ value±SD, i.e. as the concentration capable of inhibiting 50% ofthe specific radioligand-receptor binding. The IC₅₀ parameter waselaborated using “ALLFIT” software.

Results

The following tables give the results of the affinity ofcamptothecin-RGD conjugates and RGD peptides for vitronectin α_(v)β₃ andα_(v)β₅ receptors. The conjugates showed a potent affinity on bothintegrin receptors comparable to that observed with RGD peptides.

TABLE 1 Affinity of camptothecin-RGD conjugates for vitronectin α_(v)β₃and α_(v)β₅ receptors α_(v)β₃ α_(v)β₅ Compound IC₅₀ ± DS (nM) ST267047.7 ± 0.9 74 ± 0.8 ST2671 22.8 ± 1.2 54.2 ± 0.5  

TABLE 2 Affinity of RGD peptides for vitronectin α_(v)β₃ and α_(v)β₅receptors α_(v)β₃ α_(v)β₅ Compound IC₅₀ ± SD (nM) ST2581  1.7 ± 0.1 3.4± 0.1 ST2650 28.6 ± 0.7 0.17 ± 0.01 ST2700  7.2 ± 0.07  0.9 ± 0.005ST2649 37.6 ± 0.9  5.1 ± 0.07 ST2701 36.7 ± 0.7 2.9 ± 0.1Cytotoxicity of the Conjugates on Different Tumor Cell Lines

To evaluate the effect of the compound on survival cells, thesulphorodamine B test was used. To test the effects of the compounds oncell growth, PC3 human prostate carcinoma, A498 human renal carcinoma,A2780 human ovarian carcinoma cells and NCI-H460 non-small cell lungcarcinoma were used. A2780, NCI-H460 and PC3 tumor cells were grown RPMI1640 containing 10% fetal bovine serum (GIBCO), whereas A498 tumor cellswere grown in EMEM containing 10% fetal bovine serum (GIBCO).

Tumor cells were seeded in 96-well tissue culture plates (Corning) atapproximately 10% confluence and were allowed to attach and recover forat least 24 h. Varying concentrations of the drugs were then added toeach well to calculate their IC50 value (the concentration whichinhibits the 50% of cell survival). The plates were incubated for 72 hat 37° C. or 2 h followed by 72 h of recovery. At the end of thetreatment, the plates were washed by removal of the surnatant andaddition of PBS 3 times. 200 μl PBS and 50 μl of cold 80% TCA wereadded. The plates were incubated on ice for at least 1 h. TCA wasremoved, the plates were washed 3 times for immersion in distilled-waterand dried on paper and at 40° C. for 5 min.

Then 200 μl of 0.4% sulphorodamine B in 1% acetic acid were added. Theplates were incubated at room temperature for other 30 min.Sulphorodamine B was removed, the plates were washed for immersion in 1%acetic acid for 3 times, then they were dried on paper and at 40° C. for5 min.

Then 200 μl Tris 10 mM were added, the plates were kept under stirringfor 20 min. The survival cell was determined as optical density by aMultiskan spectrofluorimeter at 540 nm. The amount of cells killed wascalculated as the percentage decrease in sulphorodamine B bindingcompared with control cultures.

The IC₅₀ values were calculated with the “ALLFIT” program.

The conjugate ST2670 showed the most potent cytotoxic activity on A2780ovarian tumor cells with an IC50 value of 0.4 μM. Moreover, theconjugate ST2670 showed a comparable cytotoxicity to that observed withST2677 (free camptothecin) on tumor cells (Table 3). The conjugateST2671 also revealed a potent cytotoxicity on PC3 tumor cells comparableto that found with the free camptothecin ST2676.

TABLE 3 Cytotoxicity of the conjugates ST2670 and ST2671 and of the freecamptothecins (ST2676 and ST2677) on PC3, A498 and A2780 tumor cells (72h of treatment) PC3 A498 A2780 Compound IC50 ± SD, μM ST2670 9.6 ± 0.61.6 ± 0.3  0.4 ± 0.05 ST2671 0.35 ± 0.08 n.d. n.d. ST2676 0.038 ± 0.0040.047 ± 0.005 <0.00097 ST2677 5.42 ± 0.55 1.37 ± 0.3  0.079 ± 0.003 n.d.= not determined.

TABLE 4 Cytotoxicity of the free camptothecins 20-O derivatives (ST2676,ST2677, ST2678) on H460 non-small cell lung carcinoma cells (2 h oftreatment) NCI-H460 Compound IC₅₀ ± SD, μM ST2676 0.17 ± 0.02 ST2677 >1ST2678 0.025 ± 0.002

1. Compounds of Formula (I)

where: R₁ is the U-X—Y group, in which: U is one of the following groups—COCHR₁₀NH— or CON[(CH₂)_(n2)NHR₇]—CH₂—, where R₁₀ is H or is selectedfrom the group consisting of: linear or branched C₁-C₄ alkyl, optionallysubstituted with C₆-C₁₄ aryl or an amino-alkyl C₁-C₄; R₇ is H or linearor branched C₁-C₄ alkyl; n₂ is an integer number from 2 to 6; X is H oris a group selected among the following: —COCHR₃NH—,—COCHR₆(CH₂)_(n3)R₄—, —R₄—CH₂(OCH₂CH₂)_(n4)OCH₂R₄—, —R₄(Q)R₄—,—R₅[Arg-NH(CH₂)_(n5)CO]_(n6)R₅—, —R₅—[N-guanidinopropyl-Gly]_(n6)R₅—, inwhich n₃ is an integer number from 0 to 5, n₄ is an integer number from0 to 50, n₅ is an integer number from 2 to 6, n₆ is an integer numberfrom 2 to 7; R₃ is H or linear or branched C₁-C₄ alkyl, optionallysubstituted with —COOH, —CONH₂, —NH₂ or —OH; R₄ is selected from thegroup consisting of: —NH—, —CO—, —CONH—, —NHCO—; R₅ is either absent oris the group —R₄(Q)R₄—; R₆ is H or NH₂; Q is selected from the groupconsisting of: linear or branched C₁-C₆ alkylene; linear or branchedC₃-C₁₀ cycloalkylene; linear or branched C₂-C₆ alkenylene; linear orbranched C₃-C₁₀ cyclo-alkenylene; C₆-C₁₄ arylene; arylene(C₆-C₁₄)-alkylene; (C₁-C₆), alkylene (C₁ -C₆)-arylene (C₆-C₁₄); aromaticor non-aromatic heterocyclyl (C₃-C₁₄), containing at least oneheteroatom selected from the group consisting of O, N, S; Y is absent orH or is the following group c(Arg-Gly-Asp-AA₁-AA₂), in which: c meanscyclic; AA₁ is selected from the group consisting of: (D)-Phe, (D)-Trp,(D)-Tyr, (D)-2-naphthylAla, (D)-4-terbutyl-Phe, (D)-4,4′-biphenyl-Ala,(D)-4-CF₃-Phe, (D)-4-acetylamine-Phe; AA₂ is selected from the groupconsisting of: NW—CH[(CH₂)_(n7)—CO]—CO, NW—CH[(CH₂)_(n7)—NH]—CO,NW—[4—(CH₂)_(n7)—CO]-Phe, NW—[4—(CH₂)_(n7)—NH]-Phe, [NW]-Gly, NW-Val, inwhich W is selected from H, linear or branched C₁-C₆ alkyl,—(CH₂)_(n7)—COOH where n₇ is an integer number from 0 to 5,4-carboxybenzyl, 4-aminomethylbenzyl; the N₁-oxides, racemic mixtures,their single enantiomers, their single diastereoisomers, the forms E andZ, mixtures thereof, and their pharmaceutically acceptable salts.
 2. Acompound according to claim 1, having the following formula:

the N₁-oxides, racemic mixtures, their single enantiomers, their singlediastereoisomers, the forms E and Z, mixtures thereof, and itspharmaceutically acceptable salts.
 3. A compound according to claim 1,having the following formula:

the N₁-oxides, racemic mixtures, their single enantiomers, their singlediastereoisomers, the forms E and Z, mixtures thereof, itspharmaceutically acceptable salts.
 4. A pharmaceutical compositioncontaining at least one compound according to claim 1 as the activeingredient in a mixture with at least one pharmaceutically acceptableexcipient and/or vehicle.
 5. A method of treating non-small cell lungcancer, prostate cancer, ovarian carcinoma, or renal carcinoma,comprising administering to a subject an effective amount of a compoundof claim
 1. 6. Process for the preparation of compounds of Formula I

where: R₁ is the U—X—Y group, in which: U is one of the following groups—COCHR₁₀NH— or CON[(CH₂)_(n2)NHR₇]—CH₂—, where R₁₀ is H or is selectedfrom the group consisting of: linear or branched C₁-C₄ alkyl, optionallysubstituted with C₆-C₁₄ aryl or an amino-alkyl C₁-C₄; R₇ is H or linearor branched C₁-C₄ alkyl; n₂ is an integer number from 2 to 6; X is H oris a group selected among the following: —COCHR₃NH—,—COCHR₆(CH₂)_(n3)R₄—, —R₄—CH₂(OCH₂CH₂)_(n4)OCH₂R₄—, —R₄(Q)R₄—, —R₅[Arg-NH(CH₂)_(n5)CO]_(n6)R₅—, —R₅-[N-guanidinopropyl-Gly]_(n6)R₅—, in whichn₃ is an integer number from 0 to 5, n₄ is an integer number from 0 to50, n₅ is an integer number from 2 to 6, n₆ is an integer number from 2to 7; R₃ is H or linear or branched C₁-C₄ alkyl, optionally substitutedwith —COOH, —CONH₂, —NH₂ or —OH; R₄ is selected from the groupconsisting of: —NH—, —CO—, —CONH—, —NHCO—; R₅ is either absent or is thegroup —R₄(Q)R₄—; R₆ is H or NH₂; Q is selected from the group consistingof: linear or branched C₁-C₆ alkylene; linear or branched C₃-C₁₀cycloalkylene; linear or branched C₂-C₆ alkenylene; linear or branchedC₃-C₁₀ cyclo-alkenylene; C₆-C₁₄arylene; arylene (C₆-C₁₄)-alkylene;(C₁-C₆), alkylene (C₁-C₆)-arylene (C₆-C₁₄); aromatic or non-aromaticheterocyclyl (C₃-C₁₄), containing at least one heteroatom selected fromthe group consisting of O, N, S; Y is absent or H or is the followinggroup c(Arg-Gly-Asp-AA₁-AA₂), in which: c means cyclic; AA₁ is selectedfrom the group consisting of: (D)-Phe, (D)-Trp, (D)-Tyr, (D)-2-naphthylAla, (D)-4-terbutyl-Phe, (D)-4,4′-biphenyl-Ala, (D)-4-CF₃-Phe,(D)-4-acetylamine-Phe; AA₂ is selected from the group consisting of:NW—CH[(CH₂)_(n7)—CO]CO, NW—CH[(CH₂)_(n7)—NH]—CO,NW—[4—(CH₂)_(n7)—CO]-Phe, NW—[4—(CH₂)_(n7)—NH]-Phe, [NW]-Gly, NW-Val, inwhich W is selected from H, linear or branched C₁-C₆ alkyl,—(CH₂)_(n7)—COOH where n₇ is an integer number from 0 to 5,4-carboxybenzyl, 4-aminomethylbenzyl; the N₁-oxides, racemic mixtures,its single enantiomers, its single diastereoisomers, the forms E and Z,mixtures thereof, and its pharmaceutically acceptable salts; carried outaccording to the following reaction scheme:7-t-but-CP+U₁—X₁—Y₁ where 7-t-but-CP represents7-t-butoxyiminomethylcamptothecin, U₁, X₁ and Y₁ represent respectivelythe groups U, X and Y as defined above.
 7. Process for the preparationof compounds of Formula I

where: R₁ is the U-X-Y group, in which: U is one of the following groups—COCHR₁₀NH—or CON[(CH₂)_(n2)NHR₇]—CH₂—, where R₁₀ is H or is selectedfrom the group consisting of: linear or branched C₁-C₄ alkyl, optionallysubstituted with C₆-C₁₄ aryl or an amino-alkyl C₁-C₄; R₇ is H or linearor branched C₁-C₄ alkyl; n₂ is an integer number from 2 to 6; X is H oris a group selected among the following: —COCHR₃NH—,—COCHR₆(CH₂)_(n3)R₄—, —R₄—CH₂(OCH₂CH₂)_(n4)OCH₂R₄—, —R₄(Q)R₄—, —R₅[Arg-NH(CH₂)_(n5)CO]_(n6)R₅—, —R₅—[N-guanidinopropyl-Gly]_(n6)R₅—, in whichn₃ is an integer number from 0 to 5,n₄ is an integer number from 0 to50, n₅ is an integer number from 2 to 6, n₆ is an integer number from 2to 7; R₃ is H or linear or branched C₁-C₄ alkyl, optionally substitutedwith —COOH, —CONH₂,—NH₂ or —OH; R₄ is selected from the group consistingof: —NH—, —CO—, —CONH—, —NHCO—; R₅ is either absent or is the group—R₄(Q)R₄—; R₆ is H or NH_(2;) Q is selected from the group consistingof: linear or branched C₁-C₆ alkylene; linear or branched C₃-C₁₀cycloalkylene; linear or branched C₂-C₆ alkenylene; linear or branchedC₃-C₁₀ cyclo-alkenylene; C₆-C₁₄ arylene; arylene (C₆-C₁₄)-alkylene;(C₁-C₆), alkylene (C₁-C₆)-arylene (C₆-C₁₄); aromatic or non-aromaticheterocyclyl (C₃-C₁₄), containing at least one heteroatom selected fromthe group consisting of O, N, S; Y is absent or H or is the followinggroup c(Arg-Gly-Asp-AA₁-AA₂), in which: c means cyclic; AA₁ is selectedfrom the group consisting of: (D)-Phe, (D)-Trp, (D)-Tyr,(D)-2-naphthylAla, (D)-4-terbutyl-Phe, (D)-4,4′-biphenyl-Ala,(D)-4-CF₃-Phe, (D)-4-acetylamine-Phe; AA₂ is selected from the groupconsisting of: NW—CH[(CH₂)_(n7)—CO]—CO, NW—CH[(CH₂)_(n7)—NH]—CO,NW—[4—(CH₂)_(n7)—CO]-Phe, NW—[4—(CH₂)_(n7)—NH]-Phe, [NW]-Gly, NW-Val, inwhich W is selected from H, linear or branched C₁-C₆ alkyl,—(CH₂)_(n7)—COOH where n₇ is an integer number from 0 to 5,4-carboxybenzyl, 4-aminomethylbenzyl; the N₁-oxides, racemic mixtures,its single enantiomers, its single diastereoisomers, the forms E and Z,mixtures thereof, and its pharmaceutically acceptable salts; carried outaccording to the following reaction scheme:7-t-but-CP-U₁+X₁−Y₁ where 7-t-but-CP represents7-t-butoxyiminomethylcamptothecin, U₁, X₁ and Y₁ represent respectivelythe groups U, X and Y as defined above.
 8. Process for the preparationof compounds of Formula I

where: R₁ is the U—X—Y group, in which: U is one of the following groups—COCHR₁₀NH—or CON[(CH₂)_(n2)NHR₇]—CH₂—, where R₁₀ is H or is selectedfrom the group consisting of: linear or branched C₁-C₄ alkyl, optionallysubstituted with C₆-C₁₄ aryl or an amino-alkyl C₁-C₄; R₇ is H or linearor branched C₁-C₄ alkyl; n₂ is an integer number from 2 to 6; X is H oris a group selected among the following: —COCHR₃NH—,—COCHR₆(CH₂)_(n3)R₄—, —R₄—CH₂(OCH₂CH₂)_(n4)OCH₂R₄—, —R₄(Q)R₄—, —R₅[Arg-NH(CH₂)_(n5)CO]_(n6)R₅—, —R₅—[N-guanidinopropyl-Gly]_(n6)R₅—, in whichn₃ is an integer number from 0 to 5, n₄ is an integer number from 0 to50, n₅ is an integer number from 2 to 6, n₆ is an integer number from 2to 7; R₃ is H or linear or branched C₁-C₄ alkyl, optionally substitutedwith —COOH, —CONH₂, —NH₂ or —OH; R₄ is selected from the groupconsisting of: —NH—, —CO—, —CONH—, —NHCO—; R₅ is either absent or is thegroup —R₄(Q)R₄—; R₆ is H or NH₂; Q is selected from the group consistingof: linear or branched C₁-C₆ alkylene; linear or branched C₃-C₁₀cycloalkylene; linear or branched C₂-C₆ alkenylene; linear or branchedC₃-C₁₀ cyclo-alkenylene; C₆-C₁₄ arylene; arylene (C₆-C₁₄)-alkylene; (C₁-C₆), alkylene (C₁ -C₆)-arylene (C₆-C₁₄); aromatic or non-aromaticheterocyclyl (C₃-C₁₄), containing at least one heteroatom selected fromthe group consisting of O, N, S; Y is absent or H or is the followinggroup c(Arg-Gly-Asp-AA₁-AA₂), in which: c means cyclic; AA₁ is selectedfrom the group consisting of: (D)-Phe, (D)-Trp, (D)-Tyr, (D)-2-naphthylAla, (D)-4-terbutyl-Phe, (D)-4,4′-biphenyl-Ala, (D)-4-CF₃-Phe,(D)-4-acetylamine-Phe; AA₂ is selected from the group consisting of:NW—CH[(CH₂)_(n7)—CO]—CO, NW—CH [(CH₂)_(n7)—NH]—CO,NW—[4—(CH₂)_(n7)—CO]-Phe, NW—[4—(CH₂)_(n7)—NH]-Phe, [NW]-Gly, NW-Val, inwhich W is selected from H, linear or branched C₁-C₆ alkyl,—(CH₂)_(n7)—COOH where n₇ is an integer number from 0 to 5,4-carboxybenzyl, 4-aminomethylbenzyl; the N₁-oxides, racemic mixtures,its single enantiomers, its single diastereoisomers, the forms E and Z,mixtures thereof, and its pharmaceutically acceptable salts; carried outaccording to the following reaction scheme:7-t-but-CP-U₁−X₁+Y₁ where 7-t-but-CP represents7-t-butoxyiminomethylcamptothecin, U₁, X₁ and Y₁ represent respectivelythe groups U, X and Y as defined above.
 9. The compound

its N₁-oxides, racemic mixtures, its single enantiomers, its singlediastereoisomers, the forms E and Z, mixtures thereof, itspharmaceutically acceptable salts.